Therapeutic composition for autoimmune conditions

ABSTRACT

A therapeutic composition for the treatment, alleviation or prophylaxis of autoimmune conditions is described. The composition comprises an enzyme and an immunogen appropriate to the condition to be treated. The composition may be given in conventional fashion but is preferably given by intradermal injection.

This invention relates to a therapeutic composition. More particularly,the present invention relates to a therapeutic composition for thetreatment, alleviation or prophylaxis of autoimmune conditions.

Autoimmunity is present to some extent in everyone and is usuallyharmless. However, autoimmunity can cause a broad range of humanillnesses, known collectively as autoimmune diseases, disorders orconditions. Autoimmune conditions occur when there is progression frombenign autoimmunity to pathogenic autoimmunity when a misdirected immuneresponse occurs in an individual in which the immune system attacks thebody itself rather than a foreign body or a xenobiotic or other foreigncompound or moiety. This progression is determined by genetic influencesas well as by environmental triggers.

Immune reactions are nearly always characterised by inflammation, whichindicates an underlying repair process. However, in the case ofautoimmune conditions or diseases the inflammation may be chronic,causing tissue damage. For example, in rheumatoid arthritis chronicinflammation causes characteristic damage to joints and to cartilage.The precise origin and pathophysiological processes of these diseasesare not fully known.

Current treatments for autoimmune conditions are generally concernedwith pain management, the administration of anti-inflammatory drugs,replacing lost substance (for example, the provision of insulin indiabetes mellitus) or the administration of one or moreimmunosuppressants. These treatments are generally systemic rather thanlocal and as such may cause adverse effects elsewhere in the body.

While these approaches may temporarily alleviate the conditions, orreduce their progress, they often act to directly counteract the actualphysical state or effect rather than to remove, reduce or alter theunderlying cause or aetiology of the condition. For example, it is knownthat many autoimmune reactions involve a T-cell mediated response, itwould therefore be beneficial to provide a treatment for autoimmuneconditions which acts on this response.

Autoimmunity is evidenced by the presence of autoantibodies (antibodiesdirected against the body) and T-cells which are reactive with hostantigens. Autoimmune conditions may be systemic, for example systemiclupus erythematosus, or organ specific, for example thyroiditis. Otherexamples of autoimmune conditions include Sjögren's syndrome,Hashimoto's thyroiditis, Myasthenia gravis, rheumatoid arthritis,juvenile (type 1) diabetes, polymyositis, scleroderma, Addison'sdisease, vitiligo, pernicious anaemia, glomerulonephritis, and pulmonaryfibrosis.

It is an object of the present invention to provide a therapeuticcomposition which mediates an effect on an autoimmune condition byacting on the underlying cause or causes of the condition. However, thepresent invention may additionally have an effect on the physical stateor effect of the condition.

Accordingly, the present invention provides a therapeutic compositionfor autoimmune conditions, the composition comprising an enzyme and animmunogen at a dose which provides a beneficial effect in an individualin need of treatment.

Advantageously, the composition of the present invention mediates aresponse which acts on or affects the underlying cause of the autoimmunecondition. For example, it may act to downregulate T-cell mediatedreactions. Without wishing to be bound by theory, the present inventorbelieves that the immunogens of the present invention indirectly reduceT-cell activity by means of an action mediated via the Langerhans or thedendritic cells or by the thymus, which action redirects antigensensitive lymphocytes towards regulatory function (e.g. IL-10production) or redirects cell activity away from the target site of theimmunogen.

The term “immunogen” as used herein is intended to define any substancecapable of inducing an immune response. It is not intended that any ofthe properties of the immunogen, such as molecular weight, are to berestricted by this term.

In the description which follows, the present invention will bedescribed with particular reference to the treatment of rheumatoidarthritis. However, the invention finds equal utility in the treatmentof other disorders by the selection of an appropriate immunogen. Forexample, multiple sclerosis may be treated by the use of myelin basicprotein as the immunogen, thyroiditis or Hashimoto's disease may betreated using thyroid proteins as the immunogen, and diabetes mellitusmay be treated using insulin or β-cell proteins as the immunogen.Additionally, mixtures or combinations of immunogens may be used,especially where a condition implicates or is associated with one ormore immunogens.

Preferably, the enzyme used in the composition is a liver enzyme or amucopolysaccharidase. More preferably, the enzyme is a glucuronidase andmost preferably is a β-glucuronidase. Ideally, the p-glucuronidase isβ-D-glucuronoside glucuronosohydrolase (Registry number EC 3.2.1.31).The source of the enzyme has been found to make no difference to theactivity of the composition, provided that the enzyme is free frompreservatives or sorbitol. Hence, it may be necessary to purify theenzyme to enable its use in the composition of the invention. Any methodof purification may be used but it has been found to be convenient touse gel filtration chromatography or tangential flow filtration.

It is preferred that the enzyme is purified to a concentration of atleast 20,000 Fishman units/mg and is present in the composition at aconcentration of between 200 and 10,000 units/ml and ideally between1,000 and 5,000 units/ml.

It has been found that contamination by other proteins, even at very lowlevels can affect the activity of the enzyme. It is therefore preferredthat a stabiliser and/or activator is present in the composition. Thestabiliser and/or activator is preferably an inert proteinaceous moiety,for example protamine sulphate or 1,10 diamino decane. Preferably, thestabiliser and/or activator is present at a concentration of up to 20mg/l. Where the stabiliser and/or activator is protamine sulphate, it ispreferably present at a concentration of between 1 and 10 mg/l, morepreferably at a concentration of between 3 and 9 mg/l and ideally atabout 6 mg/l (equivalent to 6 μg/ml).

The composition may further comprise hydroxyl moieties. Preferably, thehydroxyl moieties are provided by polyols which contain at least twohydroxyl moieties and more preferably by sugars or dials which containat least two hydroxyl moieties. The preferred source of the hydroxylmoieties is 1,3 cyclohexane diol. Preferably, the 1,3 cyclohexane diolis present at a concentration of up to 20 μg/l, more preferably the 1,3cyclohexane dot is present at a concentration of between 0.1 and 10 μg/land ideally at a concentration of 1 μg/l. The stereochemistry of the 1,3cyclohexane diol has been found not to adversely affect the presentinvention and hence either the cis, or trans forms or a racemic mixturemay be used.

The composition is preferably buffered to neutral or an acid pH. Morepreferably, the composition is buffered to a pH of between 5 and 6.5 andideally the composition is buffered to pH 5.9.

In the preferred embodiment of the invention where the composition isused in the treatment of rheumatoid arthritis the preferred immunogen iscollagen or fragments, derivatives, conjugates, mimetics or otherproducts thereof or which have a collagen-type structure or activitywhether natural, synthetic or modified, regardless of source. The term“collagen” as used hereafter is intended to include such collagenproducts as above described. The collagen is preferably present in asolution. The collagen may be from any source but it is preferred thatthe collagen be free from preservatives or sorbitol or other additives.Hence, it may be necessary to purify the collagen to enable its use inthe composition of the invention. Any method of purification may be usedbut it has been found to be convenient to use gel filtrationchromatography or tangential flow filtration.

The concentration of collagen present in the composition may be ofbetween 10 and 1×10¹⁵ molecules/ml. More preferably, the collagenpresent in the composition may be at a concentration of between 1×10⁴and 1×10¹³ molecules/ml. Generally, the concentration of the collagenpresent in the composition will vary according to the dose required, itis therefore contemplated that three ranges of collagen dosedcompositions will be made available, these will be vary in strength fromhigh to low. Compositions in the high strength range will containcollagen at a concentration of the order of 1×10¹⁰ to 1×10¹⁵molecules/ml, and more preferably will contain about 1×10¹² to 1×10¹³molecules/ml. Ideally the high strength composition will contain2.5×10¹³ molecules/ml. For compositions in the mid-strength range,collagen will preferably be present at a concentration of the order of1×10⁹ to 1×10¹³ molecules/ml, and more preferably will contain about1×10¹⁰ to 1×10¹² molecules/ml. Ideally the mid-strength composition willcontain 2.5×10¹¹ molecules/ml. For compositions in the low strengthrange, collagen will preferably be present at a concentration of theorder of 1×10⁹ to 1×10¹³ molecules/ml, and more preferably will containabout 1×10¹⁰ to 1×10¹² molecules/ml. Ideally the mid-strengthcomposition will contain 2.5×10¹¹ molecules/ml.

Preferably, the composition further comprises a glycosaminoglycan ormixtures or combinations thereof. Any glycosaminoglycan can be used butit is preferred that the glycosaminoglycan be selected from the groupcomprising hyaluronate (D glucuronic acid N acetyl D glucosamine),chondroitin sulphate (D glucuronic acid N acetyl D galactosamine 1, 3, 4or 6 sulphate), dermatan sulphate (D giucuronic acid or L iduronic acidN acetyl galactosamine), keratan sulphate (D galactose N acetyl Dglucosamine sulphate), and heparan sulphate (D glucuronic acid or Liduronic acid N acetyl D glucosamine). The most preferredglycosaminoglycan is chondroitin-6-sulphate.

The glycosaminoglycan is preferably present in the composition at aconcentration of between 0.1 and 1.0 mg/ml, most preferably 0.5 mg/ml.Ideally the glycosaminoglycan is free from preservatives or sugars andto ensure this it may be necessary to purify the glycosaminoglycanbefore use. Convenient methods of purification include gel filtrationchromatography or tangential flow filtration.

The composition of the invention may be administered in any conventionalmanner either systemically or locally, for example by oral-,parenteral-, intra-dermal-, topical-, rectal-, nasal-routes, by localinjection or by transdermal infusion. At present it is preferred thatthe composition is administered by sub-cutaneous injection, preferablyby intradermal injection, or as any form of trans-dermal infusion. It isnot necessary for the composition to be administered locally to theregion of autoimmunity, especially in rheumatoid arthritis, but it maybe preferable to do so in other conditions in order to minimise anycontra-indications or to expedite an effect at a particular location.

In a preferred embodiment, the composition is prepared a short timebefore administration or even immediately prior to administration. Inthis embodiment the composition may be provided as two preparations, anenzyme preparation and a collagen preparation, which are introduced toone another and mixed prior to administration. In this embodiment, theenzyme solution contains the stabilised enzyme, the hydroxyl moiety andthe enzyme in a buffered solution; all of which are present as describedabove. The collagen solution contains the collagen and theglycosaminoglycan, buffered as described above. Preferably, thecomposition as administered contains more collagen solution than enzymesolution, more preferably at least twice the amount of collagen solution(by volume) and ideally about 4 parts collagen solution to each partenzyme solution, by volume.

Accordingly, the present invention also provides a kit for preparing thecomposition of the invention, the kit comprising an enzyme solution andan immunogen solution, the two solutions being introduced to one anotherand allowed to admix prior to administration to an individual in need oftreatment. The kit may be presented in the form of a multi-chambered ormulti-barrelled dispenser such as a syringe.

The composition of the present invention may preserved between formationand use. For example, the composition may be frozen, dried,freeze-dried, lyophilized, encapsulated or further preserved with asuitable chosen preservative which has little or no adverse effect onthe in vivo activity of the composition or by any other preservingtechnique commonly used or known for pharmaceuticals. Where thecomposition is dried or freeze-dried or otherwise rendered solid, thecomposition may be formed into a tablet, capsule, lozenge or other soliddosage form for oral administration or reconstituted for use in asolution or liquid form, for example for injection. Where thecomposition is frozen, it may be convenient to freeze the composition orits components in dose unit amounts, optionally in a syringe, tofacilitate use by the individual or medical practitioner.

Any pharmaceutically acceptable solvent may be used to produce theliquid form of the composition. Similarly, the usual binders,excipients, vehicles, and other standard dosage additives may be used inthe composition of the invention.

The present invention also provides a method of treating or preventingautoimmune conditions, the method comprising the administration of atherapeutically effective amount of a composition comprising an enzymeand an immunogen to an individual in need of treatment.

The present invention further provides a method of treating, alleviatingor preventing rheumatoid arthritis, the method comprising theadministration of a therapeutically effective amount of a compositioncomprising β-glucuronidase and collagen to an individual in need oftreatment.

The present invention also provides the use of a therapeuticallyeffective amount of an enzyme and an immunogen in the preparation of amedicament for the treatment or prevention of autoimmune conditions.

In a further aspect the present invention also provides the use of aβ-glucuronidase and collagen in the preparation of a medicament for thetreatment of rheumatoid arthritis.

In a final embodiment, the present invention provides a compositioncomprising 0.5-2.5 mg/ml β-glucuronidase, 6 μg/ml protamine sulphate, 1μg/ml 1,3 cyclohexane diol, and 0.5 mg/ml chondroitin sulphate, bufferedto pH 5.9 and further comprising either 2.5×10¹³, 2.5×10¹¹ or 2.5×10⁵molecules/ml of collagen for use in the treatment of rheumatoidarthritis.

Embodiments of the invention will now be described, by way of exampleonly, with reference to the following accompanying drawings, of which:—

FIG. 1 is a graph showing the course of arthritis in the control group(Group B).

FIG. 2 is a graph showing a comparison of the course of disease betweenGroup A and Group B over time.

FIG. 3 shows severity of arthritis in each of the experimental groups A,B and C on day 29.

FIG. 4 is a graph showing a comparison of the course of disease betweenGroup B and Group C over time.

FIG. 5 shows severity of arthritis in each of the experimental groups A,B and C on day 52 and indicates that high dose treatment significantlyreduced peak arthritis score.

EXAMPLE 1

β-glucuronidase (EC 3.2.1.31) (obtained from the marine mollusc Haliotismidae (South African abalone) was purified by gel filtrationchromatography to remove any preservatives or sorbitol present.

The purified β-glucuronidase was added to a buffered solution at pH 5.9to give a final concentration of 1.5 mg/ml. 1,3 cyclohexane diol (Sigma,Poole, Dorset, UK) was added to a final concentration of 1 μg/ml.Protamine sulphate BP was added, with stirring to prevent precipitation,to a final concentration of 6 μg/ml.

Separately, natural collagen-type II was purified by gel filtrationchromatography to remove any preservatives or sorbitol present.

The purified collagen was dissolved in a solution and buffered to pH 5.9to give a final collagen concentration of 2.5×10¹³. To this solution,chondroitin sulphate was added to give a final concentration of 0.5mg/ml.

0.01 ml of enzyme solution was introduced to 0.04 ml of collagensolution and allowed to mix. The 0.05 ml bolus of composition was usedas an intradermal injection in an arthritis model in mouse. Paw weightsand volumes were measured against control mice receiving vehicle only,collagen only or β-glucuronidase only.

EXAMPLE 2 Effects of p-Glucuronidase/Type II Collagen onCollagen-Induced Arthritis in the DBA/1 Mouse Location

The test samples were prepared by McEwen Laboratories Ltd. Pangbourne,Berkshire, England. The test facility was at the Department of Pathologyand Microbiology, School of Medical Sciences, University of Bristol,England.

Study Schedule

The study schedule was as follows:

Study initiation date 2 Feb. 2004

Assay completion date 24 Mar. 2004

Objective of Study:

The study was designed to determine the effects of two test doses oftype II collagen in combination with β-glucuronidase on the incidenceand severity of collagen-induced arthritis. Male DBA/1 mice were chosenfor the study and arthritis was initiated using type II collagen(chicken) in complete Freunds adjuvant as the initiating stimulus.Treatments were provided as separate samples of type II collagen andβ-glucuronidase. Samples were kept at 4° C. and were mixed immediatelyprior to injection. Treatment was given as a single dose injectedsubcutaneously into the scruff of the neck on day 10 post-Induction.Animals were scored for clinical arthritis from day 17 to day 52 twiceweekly by observation of joint redness and swelling. The study centrewas blinded.

Materials and Methods Mice

Thirty male DBA/1 mice were obtained from Harlan Olac at 6 weeks of age.The animals were maintained in the animal house of The School of MedicalSciences, University of Bristol, until they had reached 12 weeks of agebefore the study was initiated. On day 0 all mice were given 100 μgchicken type II collagen emulsified into complete Freund's adjuvant(CII/CFA) by injection at the base of the tail. Treatments were given bysubcutaneous injection to the scruff of the neck on day 10 and thenclinical joint swelling was scored twice weekly from day 17 to 52.

Treatment

β-glucuronidase (E.G. 3.2.1.31) (obtained from the marine molluscHaliotis midae) was provided as a freeze-dried powder. This was furtherpurified (to remove any sorbitol and salts used in the freeze dryingprocess or as stabilisers) by size exclusion (gel filtration)chromatography and diluted to a concentration of 2 mg/ml in a bufferpH5.9. To this solution was added 1×10⁻⁸ mg/ml of 1,3-cyclohexane dialand 6×10⁻⁵ mg/ml of protamine sulphate. This solution was thenaseptically filled into vials and stored between 2° C. and 8° C. untiluse.

Type II collagen (obtained from chicken cartilage) was provided as afreeze-dried powder and was further purified as above by gel filtrationchromatography. This was then diluted in a buffer solution pH5.9containing 0.5 mg/ml chondroitin sulphate (purified from sharks'cartilage). Two dilutions were used representing approximately 50 ng/mland 50 fg/ml and these were separately dispensed aseptically into vialsand stored at 2° C. to 8° C. until use.

Treatments were provided by McEwen Laboratories Ltd and labelled asfollows.

1. Enzyme A β-glucuronidase solution as described above 2. CII ACollagen 50 fg/ml as described above 3. Enzyme B Buffer control 4. CII BBuffer control 5. Enzyme C β-glucuronidase solution as described above6. CII C Collagen 50 ng/ml as described above

The identity of the samples was recorded by McEwen Laboratories but notdisclosed to the University of Bristol in advance of the end of the inlife phase. A, B and C samples were mixed in a 1 ml syringe no longerthan 10 minutes prior to injection of 200 μl into each animal. Sampleswere stored at 4° C. until use, and kept on ice following removal fromthe refrigerator and up to the point of injection.

Experimental Groups (n=10/group)

Group A: day 0 CII/CFA, day 10 treatment with mixture A

Group B: day 0 CII/CFA, day 10 treatment with mixture B

Group C: day 0 CII/CFA, day 10 treatment with mixture C

Endpoints

Clinical score of joint swelling. Animals were inspected twice weeklyfrom day 17 to day 52. On each occasion, each of the four limbs wasgiven a score according to (0=normal; 1=slight swelling of whole jointor individual digit inflammation; 2=intermediate swelling of whole jointwith redness and/or inflammation in more than one digit; 3=moderatejoint inflammation and redness spreading to multiple digits, some signsof bone remodelling; 4=severe joint inflammation and redness spreadingto multiple digits, overt signs of bone remodelling.

Results

Sample decoding. The identity of the treatment samples was revealed tothe study centre following necropsy.

Disease was present in the control group (B) with the expected incidenceand severity for this model. Disease was present at very low level onthe first day of inspection, day 17, but the incidence and severityincreased from that day until the end of the in-life phase (FIG. 1).This progression is in keeping with the expected course of arthritis inthis model. The overall disease levels in the control group were severecompared to many similar experiments carried out in the test facility.

Disease in the low dose treatment group was delayed in onset compared tothe controls (FIG. 2). This meant that a single point T test revealed asignificant difference between severity of arthritis between Group A andGroup B as tested on day 29 (FIG. 3). Thereafter, disease severityincreased in Group A. Although it remained reduced when compared toGroup B for the duration of the experiment, the overall curve indicatedno significant effect of treatment beyond day 29.

Disease in the high dose treatment group was lower than that in Group Bfrom day 25 to the end of the experiment (FIG. 4). Disease in Group Cdid not follow the normal course for arthritis in this model, diseaseaffected a lower than normal number of animals for the majority of theexperiment, and the severity of the group as a whole was very much lowerthan expected. With the exception of two animals, disease severity inGroup C remained extremely low. The lack of protection in these twoanimals meant that the variance in the Group C data set was relativelyhigh. However, the severity of arthritis was significantly lower whencomparing Group C to Group B on days 42, 46 and 52 (Mann-Whitney Utest). The data for day 52 (as the day on which highest disease wasscored) are shown in FIG. 5. In addition, Annova analysis with KruskalWallis post-test reveals an overall significant difference in the levelof disease in Group C compared to Group B (p=0.0196).

Conclusions

Both treatment doses altered the course of arthritis in the experiment.The much less marked reduction, which appeared as a delay onprogression, with low dose treatment was significant within theexperiment. The alteration to the course of disease observed followinghigh dose treatment is suggestive of a potent anti-arthritic effect. Thelevels of disease reduction in Group C are within the range seen whenestablished anti-arthritic drugs are given during the course of similarexperiments. The fact that this level of protection was observedfollowing a single treatment is highly encouraging. Improvement oftreatment levels with the low dose may be achieved by given furtherdoses as disease progresses.

1. A therapeutic composition for the treatment of autoimmune conditions,wherein the composition comprises purified β-glucuronidase and purifiedcollagen, wherein the composition is at a dose that provides abeneficial effect to an individual in need of treatment of an autoimmunecondition.
 2. (canceled)
 3. (canceled)
 4. The composition of claim 1,wherein the β-glucuronidase is β-D-glucuronoside glucuronosohydrolase(EC 3.2.1.31).
 5. The composition of claim 1, wherein theβ-glucuronidase is present at a concentration of between 200 and 10,000Fishman units/ml.
 6. The composition of claim 1, wherein theβ-glucuronidase is present at a concentration of between 0.5 and 2.5mg/ml.
 7. The composition of claim 1, wherein the composition furthercomprises a stabiliser and/or activator.
 8. The composition of claim 7,wherein the stabiliser and/or activator is an inert proteinaceousmoiety.
 9. The composition of claim 7, wherein the stabiliser and/oractivator is selected from the group consisting of protamine sulphateand 1,10 diamino decane.
 10. The composition of claim 7, wherein thestabiliser and/or activator is present at a concentration of up to 20mg/l.
 11. A therapeutic composition for the treatment of autoimmuneconditions, the composition comprising β-glucuronidase, purifiedcollagen and a stabiliser and/or activator, wherein the composition isat a dose that provides a beneficial effect to an individual in need oftreatment of an autoimmune condition, wherein the composition furthercomprises hydroxyl moieties.
 12. The composition of claim 11, whereinthe hydroxyl moieties are provided by sugars or diols.
 13. Thecomposition of claim 11, wherein the hydroxyl moieties are provided by1,3 cyclohexane diol.
 14. The composition of claim 11, wherein thehydroxyl moieties are present at a concentration of up to 20 μg/l. 15.The composition of claim 1 or claim 11, wherein the composition isbuffered to an acid or neutral pH.
 16. The composition of claim 15,wherein the composition is buffered to a pH of between 5 and
 6. 17.(canceled)
 18. The composition of claim 11, wherein the collagen ispresent at a concentration of between 10 and 1×10¹⁵ molecules/ml. 19.The composition of claim 1, wherein the composition further comprises aglycosaminoglycan.
 20. The composition according to claim 19, whereinthe glycosaminoglycan is selected from the group consisting ofhyaluronate (D glucuronic acid N acetyl D glucosamine), chondroitinsulphate (D glucuronic acid N acetyl D galactosamine 4 or 6 sulphate),dermatan sulphate (D glucuronic acid or L iduronic acid N acetyl Dgalactosamine), keratan sulphate (D galactose N acetyl D glucosaminesulphate), and heparan sulphate (D glucuronic acid or L iduronic acid Nacetyl D glucosamine).
 21. The composition of claim 19, wherein theglycosaminoglycan is chondroitin-6-sulphate.
 22. The composition ofclaim 19, wherein the glycosaminoglycan is present at a concentration ofbetween 0.1 and 1.0 mg/ml.
 23. The composition of claim 1, wherein thecomposition is in a formulation suitable for transdermal infusion orintradermal injection.
 24. A kit for preparing the composition of claim1, wherein the kit comprises an enzyme solution and an immunogensolution, and the two solutions are introduced to one another andallowed to admix prior to administration to an individual in need oftreatment.
 25. A method of treating autoimmune conditions, the methodcomprising administering a therapeutically effective amount of thecomposition of claim 1 to an individual in need of treatment of anautoimmune condition.
 26. (canceled)
 27. (canceled)
 28. (canceled) 29.The composition of claim 1 comprising 1,000 to 5,000 Fishman units/ml ofpurified β-glucuronidase, 6 μg/ml protamine sulphate, 1 μg/ml 1,3cyclohexane diol, and 0.5 mg/ml chondroitin sulphate, buffered to pH 5.9and a concentration of purified collagen selected from the groupconsisting of 2.5×10¹², 2.5×10¹⁰ and 2.5×10⁴ molecules/ml for use in thetreatment of multiple sclerosis.
 30. The composition of claim 1, whereinthe autoimmune condition is selected from the group consisting ofrheumatoid arthritis and multiple sclerosis.
 31. The method of claim 25,wherein the autoimmune condition is selected from the group consistingof rheumatoid arthritis and multiple sclerosis.